Oral L-serine supplementation reduces production of neurotoxic deoxysphingolipids in mice and humans with hereditary sensory autonomic neuropathy type 1

Abstract

Hereditary sensory and autonomic neuropathy type 1 (HSAN1) causes sensory loss that predominantly affects the lower limbs, often preceded by hyperpathia and spontaneous shooting or lancinating pain. It is caused by several missense mutations in the genes encoding 2 of the 3 subunits of the enzyme serine palmitoyltransferase (SPT). The mutant forms of the enzyme show a shift from their canonical substrate L-serine to the alternative substrate L-alanine. This shift leads to increased formation of neurotoxic deoxysphingolipids (dSLs). Our initial analysis showed that in HEK cells transfected with SPTLC1 mutants, dSL generation was modulated in vitro in the presence of various amino acids. We therefore examined whether in vivo specific amino acid substrate supplementation influenced dSL levels and disease severity in HSAN1. In mice bearing a transgene expressing the C133W SPTLC1 mutant linked to HSAN1, a 10% L-serine–enriched diet reduced dSL levels. L-serine supplementation also improved measures of motor and sensory performance as well as measures of male fertility. In contrast, a 10% L-alanine–enriched diet increased dSL levels and led to severe peripheral neuropathy. In a pilot study with 14 HSAN1 patients, L-serine supplementation similarly reduced dSL levels. These observations support the hypothesis that an altered substrate selectivity of the mutant SPT is key to the pathophysiology of HSAN1 and raise the prospect of l-serine supplementation as a first treatment option for this disorder.

Figures

Figure 1. Sphingolipid de novo synthesis is initiated by the condensation of an activated fatty acid (normally palmitoyl-CoA) and l-serine to form SA.

This reaction is catalyzed by SPT. In HSAN1, the SPT shows a shift in substrate specificity to alanine, which results in the formation of an atypical class of dSLs. For SA and doxSA (1-deoxysphinganine), the number of hydroxyl groups is indicated by m (mono-) and d (di-), followed by the number of carbons; the second number indicates the double bonds.

Figure 2. Kinetic characterization of WT and mutant SPT.

(A) Suppression of doxSA production in SPTLC1 WT HEK cells by increasing l-serine medium concentrations in the background of varying l-alanine concentrations. (B) Suppression of doxSA generation in SPTLC1 WT and HSAN1 mutant–expressing HEK293 cells at increasing l-serine medium concentrations (constant background of 2 mM l-alanine).

Figure 3. Effects of supplementation on l-serine/l-alanine concentrations.

C133W transgenic mice were fed diets with 10% l-serine, 10% l-alanine, or were left untreated. (A) Plasma levels after 3 days on diet demonstrated a significant increase in the ratio of l-serine to l-alanine in plasma of l-serine–fed mice relative to the untreated group. In contrast, mice fed l-alanine had a decrease in amino acid ratio. (B) Sciatic nerve showed a similar change in the l-serine/l-alanine ratio after 9–12 months of supplementation. (C) Liver did not show significant differences between treatment groups. *P < 0.05; **P < 0.01. Error bars represent SEM.

Figure 4. Effects of supplementation on plasma dSL levels in C133W transgenic mice.

C133W transgenic mice were fed either 10% l-serine–supplemented or 10% l-alanine–supplemented diet. Plasma samples were drawn every third day. Lipids were extracted and subjected to acid, and base hydrolysis and free sphingoid bases were quantified by liquid chromatography–mass spectrometry. (A) SA levels did not differ as a consequence of supplementation. (B) l-serine diet induced a significant reduction in doxSA plasma levels within the first 3 days. l-alanine caused an initial increase in doxSA levels and subsequent fluctuations. (C) Dietary supplementation did not significantly affect SO plasma levels. (D) DoxSO levels decreased after l-serine supplementation, while increasing in response to l-alanine.

Figure 5. Effects of supplementation on behavior in C133W transgenic mice.

(A) Experimental design. Red bar, l-alanine supplementation; blue bars, l-serine supplementation; arrows, time of sensory testing. (B) In l-alanine–supplemented mice, a trend toward worse motor performance was seen on rotarod. (C) Mechanical sensitivity was significantly worse compared with untreated age-matched C133W transgenic and WT mice. (D) In contrast, l-serine had no effect on the motor performance of 15-month-old transgenic mice, but (E) a beneficial effect on mechanical sensitivity was seen. (F) Long-term l-serine treatment improved motor performance compared with untreated age-matched C133W transgenic and WT mice. (G) Mechanical sensitivity showed a trend toward improvement relative to untreated C133W transgenic mice. Data (mean ± SEM) show percentage of age-matched, control diet–fed WT value. **P < 0.05 vs. untreated C133W transgenic mice.

Figure 6. Effect of 9–12 months’ dietary supplementation on tissue dSL levels in C133W transgenic mice.

(A) l-serine caused a significant increase of SA in liver. (B) Compared with untreated controls, doxSA levels decreased slightly with l-serine treatment and increased with l-alanine treatment in C133W transgenic mice. (C) No significant trend was noted for SO tissue levels. (D) DoxSO was found in liver and sciatic nerve, but showed no apparent dependence upon dietary supplementation. (Because doxSO was not detected in tissue of WT mice on a control diet, mean values are expressed in pmol per μg protein). Error bars represent SEM. **P < 0.05.

Figure 7. Effects of supplementation on sciatic nerve morphology.

C133W transgenic mice were fed diets with 10% l-serine or 10% l-alanine. (A) Experimental design. Red bar, l-alanine supplementation; blue bar, l-serine supplementation; end of bar denotes time of sacrifice. (B) Quantification of unmyelinated axons revealed rescue of large-caliber axons in young l-serine–treated mice (blue) relative to age-matched untreated C133W transgenic animals (dotted black line; data from ref. 13) and young l-alanine–treated mice (red). Arrow indicates relative decrease in large-caliber axons. (C) EM images of unmyelinated axons from l-serine– and l-alanine–fed mice. Scale bars: 500 nm. (D) Myelinated axons in l-serine–treated animals demonstrated a similar trend of preservation of large axons. Dotted black line represent age-matched untreated C133W transgenic animals (data from ref. 13). Arrow indicates relative decrease in large-caliber axons. (E) EM images of myelinated axons from l-serine– and l-alanine–fed mice. Scale bars: 10 μm. (F) Change in myelination patterns after supplementation, assessed by g ratios. Dotted black line represents old untreated C133W transgenic animals (data from ref. 13). Error bars represent SEM.

Figure 8. Effects of supplementation on the reproductive system of C133W transgenic mice.

(A) Experimental design. Red bar, l-alanine supplementation; blue bar, l-serine supplementation; arrows, time of reproductive analysis. (B–D) Young C133W transgenic mice fed l-alanine–supplemented diet did not differ from C133W transgenic or WT control animals on measures of testes weight (B), sperm count (C), or sperm motility (D). (E–G) l-serine–treated C133W transgenic mice were found to have increased sperm count (F), but no differences in testes weight (E) or sperm motility (G). Data (mean ± SEM) show percentage of age-matched, control diet–fed WT value. **P < 0.05.

Figure 9. Effects of l-serine supplementation on human HSAN1 patients.

(A) Design of the pilot study. Solid line, l-serine supplementation period; gray line, washout period. Plasma levels of SA (B) and SO (D) increased substantially during l-serine treatment. In contrast, doxSA (C) and doxSO (E) decreased in HSAN1 patients, reached a nadir within 4–6 weeks, and showed greater suppression at the higher dose. During the washout phase, dSL levels began to rise. Vertical lines at 10 weeks denote the beginning of washout.